Method for selective oxidation of substituted toluenes by microbial coprinus peroxidases

ABSTRACT

The present invention relates to a process for the selective oxidation of toluene derivatives by microbial peroxidases from microorganisms of the genus  Coprinus.

[0001] The present invention relates to a process for the selective oxidation of toluene derivatives by microbial peroxidases from microorganisms of the genus Coprinus.

[0002] Substituted benzaldehydes are valuable intermediates in chemical synthesis. The chemical preparation of these benzaldehydes by oxidation of the corresponding substituted toluenes is often difficult to achieve because the methyl side chain is frequently oxidized by the oxidation reaction as far as the carboxyl function and does not remain at the stage of the aldehyde function.

[0003] Moreover, microbial oxidation processes frequently cannot be used industrially, because either they give out too small a conversion of the substituted toluenes or the oxidation proceeds as far as the benzoic acid.

[0004] It is an object of the present invention to provide a process for the selective oxidation of substituted toluenes by microbial peroxidases which both ensures a high conversion of the substrates and achieves a minimum amount of substituted benzoic acids.

[0005] We have found that this object is achieved by a process for preparing aldehydes of the formula (I) by oxidizing toluenes of the formula (II) with hydrogen peroxide in the presence of a microbial peroxidase which can be isolated from a microorganism of the genus Coprinus

[0006] where

[0007] R is C₁-C₄-alkyl, C₁-C₄-alkoxy, F, Cl, Br, I, NO₂, OH and n is 0, 1, 2.

[0008] The peroxidases which are used can be employed in various degrees of purity. A partially purified peroxidase solution which no longer has laccase activity is preferred.

[0009] The peroxidase from Coprinus spec DSM 14525 (deposited on Sep. 25, 2001 at the DSM) is particularly preferably used.

[0010] The peroxidase can be employed in a large number of solvents. Buffered solutions which comprise at least 30%, preferably at least 50%, water are preferred.

[0011] The hydrogen peroxide which brings about the oxidation can be added in stoichiometric or above-stoichiometric amounts. Continuous addition of the hydrogen peroxide to the reaction medium is preferred.

[0012] The reaction temperature can be freely chosen within wide ranges between 0 and 50° C. The enzymatic conversion is slowed down greatly at low temperatures, whereas high temperatures may lead to inactivation of the enzyme. The optimal reaction temperature is accordingly between 10 and 30° C. and can easily be established by the skilled worker by means of serial experiments.

[0013] The oxidation of the methyl side chain in the process of the invention depends on the other substituents on the aromatic nucleus and their position relative to the methyl chain. Preferred other substituents R are methyl, isopropyl, t-butyl, halogen, methoxy and nitro groups.

[0014] The position of the radical R relative to the methyl chain is evidently more crucial than the nature of the radical R. Substituents R which are ortho and para relative to the methyl chain are preferred to those in the meta position in the process of the invention. Nitrotoluenes are an exception to this. o-Nitrobenzaldehyde was obtained in a relatively low yield, whereas the m-nitrobenzaldehyde yield was comparable to p-nitrobenzaldehyde. o-Nitrotoluene was also the only substrate with which the corresponding alcohol derivative was also found. No oxidation of the methyl chain to the alcohol was observed with any of the other substrates.

[0015] The invention further relates to a microbial peroxidase from Coprinus which is able to oxidize toluene by H₂O₂ to benzaldehyde so selectively that less than 0.4 mol of benzoic acid is produced per mol of oxidized toluene.

EXAMPLE 1 Preparation of the Peroxidase

[0016]Coprinus spec. DSM 14525 was fermented in 20 l of medium (soy meal). Mycelium and soy meal were removed by centrifugation. The resulting supernatant was clarified by ultrafiltration (pore size 0.16 μm). The filtrates were then concentrated by ultrafiltration using a membrane with a 10 kDa cutoff. The peroxidase was partially purified from the filtrate by FPLC. Two chromatography steps on Q-Sepharose with pH 5 and pH 7.3 afforded a 12-fold increase in activity (specific activity was 7.6 U/mg of protein with ABTS,

[0017] 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)). The enzyme preparation had no laccase activity.

[0018] This peroxidase preparation was used for the following conversions.

EXAMPLE 2 General Method for Oxidation with Peroxidase

[0019] 10 μl of the appropriate starting compound (substituted toluene), dissolved in acetone (100 mM), was diluted in 350 μl of tartrate buffer (200 mM pH 5). 4.5 μl of hydrogen peroxide (50 mM), 45 μl of water and 25 μl of a peroxidase solution which had been prepared as in Example 1 and had an activity of 30 U/ml, i.e. 1 ml of this peroxidase solution oxidizes 30 μmol of ABTS per minute, were added.

[0020] The reaction mixtures were incubated at room temperature. 4.5 μl of hydrogen peroxide (50 mM) were added with a syringe through a membrane every 10 minutes (a total of nine times). At the half life of the biotransformation, additionally 25 μl of peroxidase solution were added. The concentration of the compounds was established by HPLC through comparison with authentic standards. The structure of the resulting compounds was confirmed by GC-MS.

[0021] The oxidations shown in Table 1 were carried out in accordance with Example 2. TABLE 1 Oxidation of various toluenes with peroxidase and hydrogen peroxide Starting compound Remaining substrate concentration Product Product concentration

0.06 mM Benzaldehyde Benzoic acid 0.26 mM 0.1 mM

<0.05 mM o-Tolualdehyde o-Toluic acid 0.57 mM 0.34 mM

<0.05 mM m-Tolualdehyde 0.05 mM

<0.05 mM p-Tolualdehyde p-Toluic acid 0.28 mM 0.15 mM

0.11 mM 3,5-Dimethylbenzaldehyde 0.1 mM

0.26 mM 2,3-Dimethylbenzaldehyde 2,3-Dimethylbenzoic acid 0.21 mM 0.15 mM

0.29 mM 2,4-Dimethylbenzaldehyde 2,5-Dimethylbenzaldehyde 3,4-Dimethylbenzaldehyde Dimethylbenzoic acid 0.33 mM 0.13 mM 0.02 mM 0.28 mM

0.19 mM 4-Isopropylbenzaldehyde 2 unidentified compounds <0.05 mM

0.34 mM 4-tert-Butylbenzaldehyde 4-tert-Butylbenzoic acid 0.06 mM 0.33 mM

<0.05 mM 4-Fluorobenzaldehyde 0.36 mM

0.2 mM 4-Bromobenzaldehyde 4-Bromobenzoic acid 0.6 mM 0.5 mM

<0.05 mM 2-Chlorobenzaldehyde 2-Chlorobenzoic acid 0.36 mM 0.31 mM

<0.05 mM 3-Chlorobenzaldehyde 3-Chlorobenzoic acid 0.13 mM 0.26 mM

<0.05 mM 4-Chlorobenzaldehyde 4-Chlorobenzoic acid 0.24 mM 0.35 mM

<0.05 mM Salicylaldehyde Salicylic acid 0.08 mM <0.05 mM

0.08 mM 3-Hydroxybenzaldehyde 3-Hydroxybenzoic acid <0.05 mM <0.05 mM

<0.05 mM 4-Hydroxybenzaldehyde 4-Hydroxybenzoic acid <0.05 mM <0.05 mM

0.19 mM p-Anisaldehyde p-Anisic acid 0.5 mM 0.2 mM

0.9 mM 3,4-Dimethoxybenzaldehyde 3,4-Dimethoxybenzoic acid 0.15 mM <0.05 mM

1.22 mM 2-Nitrobenzyl alcohol 2-Nitrobenzaldehyde 2-Nitrobenzoic acid 0.13 mM 0.05 mM <0.05 mM

1.21 mM 3-Nitrobenzaldehyde 3-Nitrobenzoic acid 0.3 mM 0.12 mM

1.1 mM 4-Nitrobenzaldehyde 4-Nitrobenzoic acid 0.3 mM 0.09 mM 

1. A process for preparing aldehydes of formula (I) comprising oxidizing toluenes of formula (II) with hydrogen peroxide in a reaction mixture in the presence of a microbial peroxidase which can be isolated from a microorganism of the genus Coprinus,

wherein R is C₁-C₄-alkyl, C₁-C₄-alkoxy, F, Cl, Br, I, NO₂, or OH, and n is 0, 1, or
 2. 2. The process of claim 1, wherein the microbial peroxidase is DSM
 14525. 3. The process of claim 1, wherein R is CH₃.
 4. A microbial peroxidase that can be derived from the genus Coprinus and is able to oxidase toluene with H₂O₂ to form a benzaldehyde, wherein less than 0.4 mol of a benzoic is produced per mol of oxidized toluene.
 5. The process of claim 2, wherein R is CH₃.
 6. The process of claim 1, wherein the microbial peroxidase has no laccase activity.
 7. The process of claim 1, wherein the hydrogen peroxide is added continuously to the reaction mixture.
 8. An aldehyde prepared by the process of claim 1 wherein R is C₁-C₄-alkyl, C₁-C₄-alkoxy, F, Cl, Br, I, NO₂, or OH, and n is 0, 1, or
 2. 9. The aldehyde of claim 8, wherein R is CH₃.
 10. The microbial peroxidase of claim 4, which is DSM
 14525. 11. The microbial peroxidase of claim 4, which has no laccase activity. 